Narrow bore porous layer open tube capillary column and uses thereof

ABSTRACT

A polymer-based PLOT capillary column prepared by in situ copolymerization of a functional monomer, which usually contains the retentive chemistries, and a crosslinking monomer, which enhances the strength of the polymer matrix, is disclosed. Also disclosed is a system comprising the polymer-based PLOT column coupled to a mass flow or concentration sensitive detector, for carrying out a chemical analysis method on samples separated by liquid chromatography using the column, and a process for using the system. Columns of the invention can be prepared in a robust fashion with a very narrow i.d., e.g., 5-15 μm. Thus, they are suitable for commercial use in ultratrace LC/MS proteomic analysis. Columns according to the invention are characterized by high resolving power and high column-to-column reproducibility. When these columns are coupled on-line with, e.g., ESI-MS detection, the resulting systems are capable of detecting the component parts of complex proteomic samples down to the low attomole to sub-attomole level.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of and claimspriority from U.S. application Ser. No. 12/306,232, filed Dec. 22, 2008,which is a 371 of International Application No. PCT/US2007/014398, filedJun. 20, 2007, which claims the priority of U.S. Provisional PatentApplication No. 60/815,314, filed on Jun. 21, 2006, the whole of both ofwhich are hereby incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Part of the work leading to this invention was carried out with UnitedStates Government support provided under a grant from the NationalInstitutes of Health, Grant No. GM-15847. Therefore, the U.S. Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

Electrospray ionization-mass spectrometry (ESI-MS) has become a routinetool in proteomic studies, primarily due to its high sensitivity, broaddynamic range, and versatility for online coupling with capillary highperformance liquid chromatography (HPLC)¹⁻³. High-resolution separationprior to MS detection allows complex mixtures to be characterized byextending both the dynamic range and detection level achievable in theanalysis. Capillary LC, using 75-150 μm i.d. reversed-phase columns,offers the advantages of high resolving power, high mass sensitivity,and low sample and mobile phase consumption, and hence are widely usedtoday. However, even with such columns, LC-MS analysis of very lowquantity samples (e.g., cells from small tissue samples obtained usinglaser capture microdissection⁴) can still be problematic. More sensitiveproteomic analysis methods are necessary to tackle many challengingbiological problems.

For a given injected sample amount, narrow-bore columns result inreduced chromatographic band dilution, the analytes being eluted in asmaller volume at a higher concentration. In addition, the volumetricflow rate is an important parameter influencing ESI sensitivity⁵⁻¹⁰. Lowflow rates resulting from the narrow bore columns lead to smallerelectrospray droplet sizes, thus enhancing analyte ionization efficiencyand reducing the effect of ion suppression, all leading to highersensitivity. Additionally, the electrospray emitter attached to such alow flow rate column can be placed nearer to the MS inlet than incomparable configurations, which improves the sampling efficiency at lowflow rates. NanoESI, at flow rates of <30 mL/min, will, thus,significantly increase the MS response compared to conventional flowrates (>300 mL/min)^(5,6,11). On the other hand, packing narrow-bore(<20 μm i.d.) columns with conventional microparticles can betechnically difficult because the decreased ratio of column i.d. toparticle size induces more frequent column clogging, and packingmicroparticles into narrow-bore (<20 μm i.d.) columns requires ultrahighpacking pressure (usually >10,000 psi) and special instrumentation.Generally, the ratio of column i.d. to particle size should be greaterthan 10 to pack dense columns reproducibly. Recently, the preparation of10 μm i.d. columns packed with 1.0 μm non-porous particles at extremelyhigh pressure has been reported¹². The back pressure of a 30 cm longcolumn can reach as high as 100,000 psi at the optimum linear velocityof 0.4 cm/s. Monolithic capillary columns are increasingly considered asa viable alternative to microparticle-packed columns because of theirmoderate back pressure and high resolving power^(6,8,13-16). It wasrecently demonstrated that low-attomole sensitivity can be achieved at aflow rate of 20 mL/min using a 20 μm i.d. PS-DVB monolithic column⁶.Even more recently, others have reported on the preparation of 20 and 10μm i.d. silica-based monolithic columns^(8,16), demonstrating sensitiveand quantitative proteomic analyses at the very low flow rate of 10nL/min¹⁶. However, in all these cases, preparation of the monolithiccolumns was difficult, in part due to the increased surface area tocolumn i.d. ratio.

Given the excellent performance of open tubular capillary gaschromatography (GC), researchers have for many years tried to implementsuch columns for LC. It was recognized early on⁴⁹ that very narrow borecolumns of 5-10 μm i.d. were necessary for open tubular LC, in order toovercome band broadening due to the laminar flow in the capillary tube.Approaches of coating the capillary tubing using silicone¹⁷ or chemicalmodification of etched surfaces¹⁸ were first developed to prepare opentubular capillary LC columns. However, such columns provided lowretention and low sample loading capacity even for small molecules, letalone for complex biological samples.

Porous layer open tube (PLOT) columns were introduced in 1960s toincrease the sample loading capacity of the GC columns¹⁹. Althoughefforts have been made in the last 20 years to prepare PLOT capillary LCcolumns²⁰⁻²³, success has been limited due in part to the following: 1)lack of a sensitive, universal, small dead volume detector²⁴; 2) lack ofability to generate effective gradient elution at very low flow rates;and 3) difficulties in the preparation of capillary columns with auniform stationary layer reproducibly. ESI-MS has proven to be an idealsensitive detector with zero dead volume, and current HPLC pumps canprovide stable flow rate at low nL/min level after accurate splitting.The remaining problem is to prepare and implement high efficiency LCPLOT columns, a major challenge being to cast a suitably uniform porouslayer on the column to provide sufficient retention and sample loadingcapacity. Several methods have been developed to realize a retentivelayer suitable for increasing the surface area and phase ratio, e.g.,static²⁵, dynamic^(26,27), and precipitation coating²⁸. To simplify thepreparation process, a method of laying down a porous siliceous layer ina single step via a sol-gel process was described²⁹. Methods ofpreparing gold nanoparticle-coated PLOT columns have also beendescribed^(30,31). However, these and other attempts³²⁻³⁶ have not beensufficiently successful to permit commercial level development of PLOTcapillary LC columns and their use, e.g., in ESI-MS.

BRIEF SUMMARY OF THE INVENTION

A new polymer-based PLOT column prepared by in situ copolymerization ofa functional monomer, which usually contains the retentive chemistries,and a crosslinking monomer, which enhances the strength of the polymermatrix, is disclosed herein. For example, styrenic based monomers suchas styrene and divinylbenzene or meth/acrylic based monomers such asbutyl or stearyl methacrylate and ethylene glycol dimethacrylate, areemployed. Columns of the invention can be prepared in a robust fashionwith a very narrow i.d., e.g., 5-15 μm. Thus, they are suitable forcommercial use in ultratrace LC/MS proteomic analysis. Columns accordingto the invention are characterized by high resolving power and highcolumn-to-column reproducibility. When these columns are coupled on-linewith, e.g., ESI-MS detection, the resulting systems according to theinvention are capable of detecting the component parts of complexproteomic samples down to the low attomole to sub-attomole level. Thepower of methods using columns of the invention is demonstrated inparticular by coupling such columns to the new mass spectrometers, suchas the hybrid linear ion-trap/FT mass spectrometer (LTQ/FT-MS,ThermoElectron, San Jose, Calif.), for bioanalyses. The high resolutionand sensitivity of these columns opens up major possibilities for thediagnosis of biopsy samples as well as the determination of specificbiomarkers that can provide molecular phenotyping of individual samples.Such developments are of clear clinical importance and therapeuticsignificance in that tissue samples of a highly limited quantity can besuccessfully analyzed for proteomic content using the columns andmethods of the invention. Also, columns according to the invention canbe online coupled to other sensitive detectors such as fluorescence,electro/chemiluminence or nuclear magnetic resonance (NMR) for, e.g.,detection of trace chemical or biological agents in chemical orbiological defense applications.

Thus, in one aspect, the invention is directed to a porous layer opentube capillary column, or channel in a microfabricated device, thecolumn or channel including a capillary column or channel having an i.d.of 15 μm or less (preferably 10 μm or less); and a rigid porous layerseparation medium comprising a highly crosslinked, macroporous, organicpolymeric stationary phase layer attached covalently to the inner wallsurface of the column or channel, wherein the organic polymericstationary phase layer includes styrenic, methacrylic or acrylicmonomeric units, or combinations thereof; wherein the organic polymericstationary phase layer is from 0.5-3 μm in thickness; wherein theorganic polymeric stationary phase layer is thermally stable to 250° C.;and wherein the reproducibility of retention time on comparable columnsor channels during use varies less than 10%, and, preferably less than5%. A preferred capillary column according to the invention has a lengthof greater than or equal to one meter, and preferably greater than orequal to three meters. In preferred embodiments of the capillary columnor channel, the organic polymeric stationary phase layer ispoly(styrene-divinylbenzene) or has (C4-C18) alkyl methacrylate monomerunits, and the column or channel during use for liquid chromatographyhas a flow rate at 6000 psi or less of 5-50 nL/min.

In another aspect, the invention is directed to method of preparing aseparation capillary column or channel in a microfabricated device, thecolumn or channel comprising a porous layer open tube separation mediumincluding a macroporous, organic polymeric stationary phase layer, saidmethod including the steps of (1) providing an unfilled capillarycolumn, or channel in a microfabricated device, the column or channelbeing open at both ends thereof and having an i.d. of 15 μm or less, theinner wall surface of the column or channel including a bifunctionalanchoring or coupling agent suitable for covalent attachment of amacroporous, organic polymeric stationary phase layer as a porous layeropen tube separation medium; (2) adding to the column or channel amixture including a functional monomer selected from the groupconsisting of styrenic, methacrylic and acrylic monomers, andcombinations thereof; a crosslinker compatible with the functionalmonomer, the crosslinker being capable of providing extensivecrosslinking; a polar porogenic solvent; and an initiator for thermal orUV induced polymerization; and (3) polymerizing the mixture in thecolumn to form the macroporous, organic polymeric stationary phase layeras the porous layer open tube separation medium attached to the innersurface of the column or channel. In preferred embodiments of the methodof the invention, the inner wall surface of the column or channel issilica and the bifunctional anchoring or coupling agent contains at oneend a functional group reactive with silica and at the other end afunctional group reactive with said functional monomer (an exemplarybifunctional anchoring or coupling agent being 3-(trimethoxysilyl)propylmethacrylate); the functional monomer in the polymerization mixture isstyrene and the crosslinking agent is divinylbenzene. In other preferredembodiments of the method, the functional monomer in said polymerizationmixture is methacrylate (e.g., (C4-C18) alkyl methacrylate, inparticular, butyl or stearyl methacrylate), and the preferredcrosslinking agent is ethylene glycol dimethacrylate. Preferredporogenic solvents include C_(n)H_(2n+1)OH, wherein 1≦n≦4), whereinethanol is particularly preferred, or acetonitrile. In other preferredembodiments of the method, the ratio of total monomer (functionalmonomer plus crosslinking monomer) to porogenic solvent in thepolymerization mixture varies between 10-40% (V/V) while the ratio offunctional monomer to crosslinking monomer varies between 1:1 to 1:3.

In another aspect, the invention is directed to a process of carryingout a chemical analysis method including the steps of providing theseparation capillary column or channel of the invention; coupling thecolumn or channel to a mass flow or concentration sensitive detector;applying an aliquot of a liquid sample to the column or channel;conducting a liquid chromatographic separation procedure on the appliedsample; and detecting the separated sample with the detector to carryout the chemical analysis method.

In yet another aspect, the invention is directed to a system forcarrying out a chemical analysis method, the system including theseparation capillary column or channel of the invention and aconcentration sensitive detector coupled with an interface to the exitend of the separation column or channel. Exemplary concentrationsensitive detectors include a mass spectrometer, a fluorescencedetector, an electro-chemiluminescence detector and a nuclear magneticresonance detector. A preferred interface is an electrospray ionization(ESI) interface or a matrix assisted laser desorption ionization (MALDI)interface. In a preferred embodiment, the system of the inventionfurther includes a preparatory precolumn coupled to the entrance end ofthe separation column or channel.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims, taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic diagram of an exemplary embodiment of a 10 μm i.d.poly(styrene-divinylbenzene) PLOT column according to the invention in amicroSPE/nanoLC/ESI-MS system according to the invention;

FIGS. 2A and 2B are scanning electron micrographs of the middle section(A) of the PLOT column of FIG. 1 and of an end section (B) of the PLOTcolumn. The end sections constitute roughly 5% of the approx. 5 m longcapillary;

FIGS. 3A-3D are MS/MS spectra from four peptides of a BSA tryptic digestwith 10 attomole injected directly onto the PLOT column;

FIGS. 4A-4E illustrate comprehensive analysis of a Lys-C digest of EGRF.(A) Base peak chromatogram from nanoLC-ESI-MS analysis of 25 fmol of aLys-C digest of EGFR injected on the 4.2 m×10 μm i.d. PS-DVB PLOT columnaccording to the invention; selected MS/MS spectra are shown for long(B), phosphorylated (C), and glycosylated (D, E) peptides of EGFR. Thepeptide sequences and the extracted ion chromatograms are shown in theinsert. The phosphothreonine is indicated as T*. The glycosylation siteis labeled N*. In the Man8 structure, the triangle (▴) and circle ()represent mannose and N-acetyl glucosamine, respectively. The sequentialloss of terminal mannoses from the Man8 structure resulted in Man7,Man6, etc. In the glycan structures, SA represents sialic acid and thesquare (▪) represents galactose; and

FIGS. 5 a and 5B are chromatograms providing for the calculation of peakcapacity for the column according to FIG. 1. FIG. 5A is the base peakchromatogram from the microSPE-nanoLC-ESI-MS analysis of a 4 ng trypticin-gel digest of a single SDS-PAGE cut of M. Acetivorans and FIG. 5Bshowns extracted ion chromatograms of six high intensity peaks used tocalculate the peak capacity.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, high-efficiency, narrow, e.g., 10 μm i.d.,PLOT columns (e.g., poly(styrene-divinylbenzene) can be repeatedlyprepared in a single copolymerization step. The polymer layer iscovalently attached to the walls of the capillary, and there is thus noneed for column frits. Column-to-column retention time reproducibilityis ˜3% RSD, and, in terms of relative retention time, ˜2% RSD. The highpermeability of the open structure allows long columns to be used atmoderate pressure, which aids sample loading capacities. Theconcentrated analyte that elutes from a PLOT column according to theinvention, combined with decreased ion suppression and enhanced ioncollection efficiency at a flow rate of, e.g., 20 nL/min, significantlyimproves ESI-MS sensitivity. Due to its open porous layer structure, thePLOT column of the invention demonstrates high efficiency for theseparation of large peptides, as well as peptides with phosphorylatedand glycosylated modifications. The columns are well suited to extendedrange proteomic analysis. The high resolution capabilities of the columnhave been demonstrated in an exemplary system described herein employingmicro-solid phase extraction, nano-liquid chromatography, electrosprayinterface, mass spectrometry (microSPE-nanoLC-ESI-MS) analysis of acomplex proteome sample using a 4.2 m×10 μm i.d. PS-DVB PLOT columncoupled with a 50 μm i.d. PS-DVB monolithic precolumn.

Preferred embodiments of the columns according to the invention usedifferent retentive chemistry functionalities compared to the prior artand a very high degree of crosslinking to prepare the inside wall layer(stationary phase) so that the stationary phase is essentially rigid.This means that there is essentially no swelling of the stationaryphase, and, consequently, no change in volume, in the presence of themobile phase, which usually contains organic solvent. These changes haveled to dramatic improvements in the resolution and reproducibility ofanalyses carried out using columns according to the invention because,without swelling of the stationary phase, the kinetics of diffusion ofthe separating components in and out of the stationary phase is morefavorable, that is, mass transfer resistance is minimized, and, thus,high performance is achieved as well as good reproducibility.

With a 10-15 μm i.d. capillary, a PLOT column having an inside walllayer thickness of ˜1-3 μm will reduce the open tube i.d. to roughly 7-8μm. This column diameter has previously been shown to be sufficient tominimize radial band broadening, leading to high performanceseparations. In addition, commercial HPLC pumps will be able to deliversufficient flow by virtue of the open tube structure. One point ofsignificance for such a column when coupled to ESI/MS is that the flowrate will be in the range of 5-50 nL/μm, more than an order of magnitudelower than results with 75 μm i.d. columns. From the early days ofnano-ESI, it has been recognized that such low flows lead to asignificant reduction in ESI droplet size such that only one ion isencapsulated in one droplet. In this case, the significant problem ofion suppression is minimized or eliminated, a feature particularlyfavorable to peptides with post-translational modifications, such ascarbohydrates or phosphates.

The reversed phase PLOT column according to the invention, e.g., 10 μmi.d., yields robust high resolution separation with minimal ionsuppression. Use of such columns would significantly impact peptidequantitation and, therefore, yield more comprehensive and accurateresults for, e.g., biomarker studies.

The invention is directed to a procedure to reproducibly prepareultra-narrow bore (i.d. <15 μm) porous layer open tube (PLOT) capillarycolumns for liquid chromatography coupled with mass spectrometry orother sensitive detection techniques such as fluorescence,electro/chemiluminence or NMR detection. The invention is also directedto the resulting columns and to their uses. In columns according to theinvention, the retentive stationary phase is a porous polymer formed by,e.g., temperature induced or UV light induced solution polymerization.

Exemplary uses of PLOT columns according to the invention includehigh-sensitive, high-efficiency gradient and isocratic single ormulti-dimensional nano-LC analysis of limited amounts of biological ormedical samples by coupling the columns at low flow rates to massflow-sensitive detectors (i.e., ESI-MS). Single, parallel or sequentialsample separation experiments using PLOT columns according to theinvention can be coupled to electrospray ionization mass spectrometry(ESI-MS) or matrix assisted laser desorption ionization massspectrometry (MALDI-MS).

One embodiment of the method according to the invention is characterizedin that the inner surface of the bare fused-silica capillary ispre-functionalized before polymerization with, e.g., an anchoringsilane, which contains acryl or methacryl groups, enabling the reactionof the anchoring silane with monomers and crosslinkers thereafter.

In another embodiment of this method, the polymerization solution iscomposed of a functional monomer, such as styrene or alkyl methacrylate;a crosslinker that provides a high degree of crosslinking, such asdivinylbenzene or ethylene glycol dimethacrylate, at a typical quantityratio of monomer/crosslinker of 1:1; and a polar porogenic solvent (orporogen), such as ethanol, methanol, propanol or acetonitrile. Theporogen chosen is one that has a negligible swelling effect on theresulting polymer formed and not one that would be a good solvent forthe resulting polymer, such as the non-polar solvents toluene,chloroform, tetrahydrofuran or heptane. The polymerization solution hasa low viscosity; thus, it can be introduced into the pre-functionalizedfused-silica capillary under low pressure, such as 100-psi or lower, fora capillary tubing length of several meters. The retentive layer thusformed using the monomers and crosslinkers described above is ready forchromatographic separation without additional surface functionalizationsteps.

Another embodiment of this method is characterized in that the polymericretentive layer, e.g., 0.5-3 μm thick, formed after polymerization isintegrated to the fused silica capillary inner wall. The layer'sstructure is rigid and characterized by a rugulose inner surface, whichenhances surface area and, thus, loading capacity.

Another embodiment of the method according to the invention ischaracterized in that no evaporation of the porogenic solution is neededafter polymerization, in contrast to other methods. The porogen issimply flushed out of the column after polymerization. Avoiding the useof a swelling porogen, such as toluene, chloroform, tetrahydrofuran,etc., which may remain in the network after polymerization and thusnecessitate an evaporation step, diminishes the problem of cloggingduring the evaporation step, thus simplifying preparation and improvingreproducibility.

In future developments, long columns up to 10 m in length areenvisioned, which can be used to improve the resolving power of thesystem further, still using a conventional LC pumping system.Furthermore, short PLOT columns run at high temperature will be usefulfor fast separation and analysis. Although, the retentive layerdescribed above can be used for chromatography separation withoutadditional surface functionalization steps (as this retentive layercontains no reactive groups, being devoid of charged functionalitiessuch as sulfonic, carboxylic, primary, secondary, tertiary andquaternary amines), PLOT columns with different surface chemistries forvarious separation modes can be easily prepared using specific monomers.For example, a more hydrophobic column could be prepared by usingstearyl methacrylate instead of styrene, or2-acrylamido-2-methyl-1-propane sulfonic acid for ion exchangechromatography. Other retentive groups, if desired, could include alkylchains, hydrophilic groups or affinity functions.

An exemplary column according to the invention is a long,high-efficiency polystyrene-divinylbenzene (PS-DVB), 10 pm i.d. porouslayer open tube (PLOT) capillary column. Repeatable PLOT capillariesaccording to the invention (˜3% RSD column-to-column), with highpermeability, were easily prepared by in-situ polymerization. Relativelyhigh loading capacities, ˜100 fmol for angiotensin I and ˜50 fmol forinsulin were obtained with a 4.2 m×10 μm i.d. PLOT column. Low detectionlevels (attomole to sub-attomole) were achieved when the column wascoupled on-line with a linear ion trap MS (LTQ). Analysis of humanepidermal growth factor receptor (EGFR), a large trans-membrane tyrosinekinase receptor with heterogeneous phosphorylation and glycosylationstructures, was obtained at the 25 fmol level. The PLOT column yielded apeak capacity of ˜400 for the separation of a 4 ng complex trypticdigest mixture when the sample preparation included a 50 μm i.d. PS-DVBmonolithic precolumn and ESI-MS detection. As an example of the power ofthe column, 3046 unique peptides covering 566 distinct Methanosarcinaacetivorans proteins were identified from a 50 ng in-gel tryptic digestsample combining five cuts in a single LC/MS/MS analysis using the LTQ.The results demonstrate the potential of the PLOT column according tothe invention for high resolution LC/MS at the ultratrace level.

The following examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These examples are not intended in any way otherwise tolimit the scope of the disclosure.

Materials

Fused silica capillary tubing with a polyimide outer coating waspurchased from Polymicro Technologies (Phoenix, Ariz.). Styrene,divinylbenzene (DVB), ethanol, formic acid (HPLC grade),3-(trimethoxysilyl)propyl methacrylate, 2,2′-diphenyl-1-picrylhydrazyl(DPPH), N,N-dimethylformamide (DMF) anhydrous, and2,2′-azobisoisobutyronitrile (AIBN) were obtained from Sigma-Aldrich(St. Louis, Mo.). Acetonitrile (HPLC grade) and deionized water (HPLCgrade) were purchased from Fisher Scientific (Fair Lawn, N.J.). Astandard tryptic digest of bovine serum albumin (BSA) was from MichromBioresources, Inc. (Auburn, Calif.). Angiotensin I, insulin from bovinepancreas, HPLC standard protein mixture (ribonuclease A (13700 Da),cytochrome C (12327 Da), apomyoglobin (17600 Da), holo-transferrin(>70000)), β-casein from milk and human epidermal growth factor receptor(EGFR) from an A431 cancer cell line were purchased from Sigma-Aldrich(St. Louis, Mo.). Achromobacter protease I (Lys-C) was obtained fromWaco Chemical Co. (Osaka, Japan), and trypsin (sequencing grade) wasfrom Promega (Madison, Wis.).

Example I Preparation and Characterization of a PLOT column According tothe Invention

Fused-silica capillary tubing with a 10 μm i.d. (˜5 meters) was firstflushed overnight with 1.0 mol/L NaOH at ˜1000 psi, washed with waterand flushed with 1.0 mol/L hydrochloric acid, and then washed again withwater and acetonitrile. The capillary was dried with nitrogen at ˜1000psi to remove residue water and acetonitrile. 30% (v/v)3-(trimethoxysilyl)propyl methacrylate and 0.5% (wt/v)2,2′-diphenyl-1-picrylhydrazyl (DPPH) in N,N-dimethylformamide anhydrous(DMF) was freshly prepared and filled into the 10 μm i.d. pretreatedcapillary. Both ends of the capillary were sealed with a septum, and thecapillary was placed in an oven at 110° C. for 6-10 h. The capillary waswashed with acetonitrile and blown dry with nitrogen at 1000 psi. Apolymerization solution was prepared containing of 5 mg of AIBN, 200 μLstyrene, 200 μL DVB, and 600 μL ethanol. The solution was degassed byultrasonication for 5 min and then filled into the silanized capillary.Both ends of the capillary were sealed with septa, and the capillary washeated at 74° C. for ˜16 h in a water bath. The column was then washedwith acetonitrile and was ready for use. In addition, 50 μm i.d. PS-DVBmonolithic precolumns were prepared using protocols describedpreviously¹⁴.

HPLC separations were performed using a Surveyor pump (ThermoElectron,San Jose, Calif.). Mobile phase A (0.1% (v/v) formic acid in water) andmobile phase B (0.1% (v/v) formic acid, 10% (v/v) water in acetonitrile)were used for the gradient separation. Samples were either bomb loadedonto the PLOT column or onto a 4 cm×50 μm i.d. PS-DVB monolithicprecolumn. A microSPE/nanoLC/ESI-MS system using a 10 μm i.d. PLOTcolumn is shown in FIG. 1. Referring now to FIG. 1, in one embodiment,samples are first loaded manually off-line onto a precolumn 10, which isthen inverted and butt-to-butt connected to a 10 μm i.d PLOT column 12using a Picoclear™ fluoropolymer core, clear elastomeric insertconnector 14 (New Objective, Woburn, Mass.). The sample is back-flushedfrom precolumn 10 onto PLOT column 12. A PEEK tee (Upchurch ScientificInc., Oak Harbor, Wash.) is used as a splitter 16, and the precolumn10/PLOT column 12 assembly is attached to arm 18 of the splitter.Gradient flow from an HPLC pump 24 is applied through in-line arm 20 ofthe splitter, and a portion of the gradient flow goes through the 90°splitting arm 22, where a 50 μm i.d. fused silica capillary can beconnected to adjust the mobile phase flow through the microSPE-LCassembly. Flow rates of the PLOT column were measured by connecting 50μm i.d. open fused-silica capillary tubing to the exit end of the PLOTcolumn, and then the volume of mobile phase that flowed for a givenperiod of time was determined.

NanoESI-MS was performed on an LCQ Deca XP or an LTQ ion trap massspectrometer (ThermoElectron). Referring again to FIG. 1, PLOT column 12was carefully butt-to-butt connected to a coated ESI spray tip 26 (360μm o.d., 20 μm i.d. fused silica with 5 μm i.d. tip, 2-3 cm in length,New Objective) using a Picoclear™ connector 14. Electrospray voltage 28was applied directly on the spray tip 26 to direct droplets of generatedsample ions to the MS inlet orifice 30. The data generated from LC/MSexperiments were analyzed using standard database searching algorithms(SEQUEST). Peptides were assigned based upon a Peptide Prophetprobability greater than 0.95, a ΔCn greater than 0.10, and Xcorrgreater than 1.8, 2.5 and 3.5 for singly, doubly and triply chargedions, respectively.

In addition to a tryptic digest sample of BSA, Lys-C digests of β-caseinand EGFR and an in-gel tryptic digest of Methanosarcina acetivorans wereused as test mixtures to evaluate the performance of the nanoLC-ESI-MS.Lys-C digestion of β-casein was performed as follows: Lys-C was spikedinto the β-casein (at 10 pmole) in a 1:40 (w/w) ratio and incubated for4 h at 37° C. (pH 8.5). For Lys-C digestion of EGFR, 10 μg lyophilizedpowder of EGFR was dissolved in 100 μL of 6 M guanidine hydrochlorideand 0.1 M ammonium bicarbonate in water. Reduction was conducted with 40mM dithiothreitol for 30 min at 37° C., followed by alkylation with 80mM of iodoacetamide for 1.5 h in the dark at room temperature. Thebuffer was subsequently exchanged to 0.1 M ammonium bicarbonate buffer,pH 8.5, to remove additional salts and reagents. Lys-C (1:20 w/w) wasadded to digest the protein for 4 h at 37° C. (pH 8.5). The mixture wasacidified with 1% formic acid to quench the digestion, followed bystorage at −20° C.

M. acetivorans cells, grown in methanol, were cultured as previouslydescribed³⁷. Protein extraction, SDS-polyacrylamide gel electrophoresis(PAGE) fractionation and in-gel digestion were performed using protocolsreported previously³⁷. The concentration of the whole-cell proteinextracts, determined by the Bradford assay (Bio-Rad, Hercules, Calif.),was 3.0 mg/mL. Roughly 45 μg of total protein was loaded on the gel, andafter electrophoresis, the gel lanes were cut into 5 fractions. Thein-gel tryptic digest of a fraction of M. acetivorans proteins (M>70kDa) was used to evaluate the performance of the PLOT column. Inaddition, all 5 in-gel digested fractions were combined together torepresent a global proteomic analysis for characterization of the PLOTcolumn.

Compared to silica-based stationary phases, organic polymeric stationaryphases provide several advantages, e.g., improved chemical stabilityover an extended pH range and the absence of silanol groups that cancause irreversible adsorption of peptides and proteins. The exemplaryPS-DVB porous layer was prepared and attached to the silanized capillarywall in a single in situ copolymerization step. Selection of a suitablesolvent for the copolymerization step is key to successful preparationof repeatable, high efficiency PLOT columns. The polymer shouldprecipitate from solution at an early stage of the polymerizationprocess, forming a thin porous layer at the capillary wall, whileleaving open the main section of the capillary tube. A porogenic solventin which the resulting polymer, e.g., PS-DVB, is not very soluble is,therefore, desirable for the preparation of the PLOT column.

Several organic solvents, including methanol, ethanol, propanol,tetrahydrofuran, and acetonitrile, were examined for their ability toprepare repeatable PLOT columns. From this group, the non-polar solventethanol was selected for further study since successful columns wereroutinely made using this solvent. The effect of the ratio of ethanol tomonomer concentration on the preparation of the PLOT column was theninvestigated. In these studies, more than 50% monomer in thepolymerization mixture was observed to lead to column blockage. At 60%ethanol/40% monomer, repeatable, high performance PLOT columns wereobtained.

The surface layer of a PLOT column, prepared from −5 meter of 10 μm i.d.fused silica capillary tubing using 60% ethanol, was then examined atdifferent sections of the column using scanning electron microscopy(SEM). Referring to FIG. 2A, it can be seen that the porous layer wasobserved to be uniform throughout most of the capillary. Portions at theends of the capillary (˜5% from each end) contained relatively largeglobules, as shown in FIG. 2B. These ends are cut to produce a column oflength ˜4.2 meters. From the SEM picture, the thickness of the surfaceinner layer of the PLOT column was estimated to be between 0.5 and 1 μm.Since there may be some globules interspersed at low density throughoutthe column, the average thickness may be somewhat higher. Earlierexperiments had suggested that the layer thickness should be in therange 0.3-2 μm for a coated open tubular column, depending on the masstransfer coefficients of the solutes between the mobile and stationaryphases⁴⁰.

Example II Characterization of Column Performance

A variety of chromatographic studies were conducted to characterize a4.2 m×10 μm i.d. PLOT column accordng to the invention. The long columnprovided a flow rate of ˜20 mL/min at pressures of only ˜2900 psi. Onthe basis of these conditions, Darcy's law⁴¹ was used to calculate thecolumn permeability as 1.3×10⁻¹² m². It is interesting to note that thisvalue is roughly 4-fold lower than an equivalent open tube capillary of10 μm i.d. without a porous layer. The lower permeability and higherpressure drop of the PLOT column according to the invention isundoubtedly due to the porous layer reducing the open tube diameter. Onthe other hand, the permeability is 15-fold higher than a recentlyintroduced 10 μm i.d. silica monolithic column¹⁶. Thus, the columnaccording to the invention is characterized by relatively highpermeability in comparison to a similarly sized packed column. Hence,very long columns according to the invention, e.g., of 4 m or greater,can be operated successfully with commercially available HPLC pumpingsystems that have a pressure limit of 6000 psi.

Use of a detection system and associated connections that make only aminimal contribution to extra column dead volume is the key to achievehigh efficiency separation of narrow bore PLOT column at such low flowrates⁴². In this study, a PicoClear union was used to connect the PLOTcolumn and the ESI emitter. Through visual inspection, the straight cutPLOT column outlet was observed to be closely connected to the coatedESI emitter, which had a 5 μm i.d. spray tip, 2-3 cm long. The emittercould be easily replaced if the tip became clogged. Stable electrospraywas readily generated from the emitter at flow rates of ˜20 mL/min.

Since the columns of the invention are made in a single step aftersilanization of the capillary wall, a simple procedure for columnproduction can be established. Reproducibility of retention from columnto column was tested in the gradient elution separation of a 1:1 mixtureof a BSA tryptic digest and a β-casein Lys-C digest. An 800-attomoleamount of the mixture was bomb loaded on the PLOT column.High-performance separation was carried out with direct loading and useof a microSPE column. Gradient: mobile phase A (0.1% (v/v) formic acidin water) to 40% B (0.1% (v/v) formic acid, 10% (v/v) water inacetonitrile) in 45 min with data collection initiated at the start ofthe gradient. Flow rate: ˜20 mL/min at an inlet pressure of ˜2900 psi.The run-to-run retention time reproducibility was established from threeindependent analyses. The consecutive run-to-run reproducibility wasfound to be better than 1.2% RSD. Three, separate PLOT columns were thenprepared, and the reproducibility of retention from column-to-column ispresented in Table 1.

TABLE 1 PLOT Column-to-Column Reproducibility^(a) Retention time (min)m/z Column 1 Column 2 Column 3 RSD, % RSD*, %^(b) 655 33.84 34.83 36.113.26 1.97 480 34.21 35.10 36.41 3.14 1.81 582 34.97 35.86 37.27 3.211.83 813 35.50 36.35 37.48 2.73 1.56 628 37.62 38.26 39.55 2.55 1.22 74139.01 39.62 40.96 2.51 1.15 879 41.57 41.93 43.36 2.24 0.77 843 42.0542.74 44.12 2.45 1.16 785 43.20 43.85 45.52 2.71 1.22 832 47.46 47.5549.42 2.30 0.50 1340 50.62 50.13 52.11 2.02 0.18 1330 52.05 51.43 53.381.91 0.34 1241 52.79 52.47 54.46 2.01 — ^(a)Mixture of BSA trypticdigest and β-casein Lys-C digest was used to test the column-to-columnreproducibility of three 4.2 m × 10 μm i.d. PS-DVB PLOT columns.^(b)RSD* represents the RSD of relative retention time normalized to theion of m/z = 1241.

It can be seen that the reproducibility for the three columns was betterthan 3% RSD, and if the retention was normalized to the ion withm/z=1241, the RSD drops to less than 2%. The results in Table 1 are verypromising given that the tube diameter was only 10 μm i.d. A likelysource of the larger column-to-column % RSD relative to that for therun-to-run retention reproducibility is minor differences in flow rateresulting from small variations in column permeability. The average peakwidth for six high-intensity m/z peaks on the three columns was alsodetermined. The peak width at half-height was 6±0.5 s on the three PLOTcolumns, again indicating good reproducibility. Finally, the PLOTcolumns showed good stability. The retention and peak widths remainedunchanged over 3 months, with hundreds of sample injections.

The loading capacities of the PLOT column were determined by measuringpeak width at half-height (w_(1/2)) as a function of injected amounts ofangiotensin I (1296.5 Da) and insulin (5733.5 Da). The maximum loadingcapacity is defined as the amount of sample injected when thecorresponding w_(1/2) is increased by 10% over the peak width at lowsample amounts. Using a fixed sample volume of 2 nL, the sample solutionat various concentrations was bomb loaded on the PLOT column.NanoLC-ESI-MS was conducted with a 20 min gradient, and the w_(1/2) foreach analysis was determined from the corresponding extracted ionchromatogram. The loading capacities of the PLOT column, prepared using60% of solvent, were ˜100 fmol for angiotensin I and ˜50 fmol forinsulin. Given that 10 μm i.d. columns were used, these values representrelatively high loading capacity.

It is useful to compare these results to the loading capacity of a 6cm×200-μm-i.d. PS-DVB monolithic column (column volume of −1.9 μL) of −1μmol, previously reported for a small peptide⁴². The column volume ofthe 4.2 m×10-μm-i.d. PLOT column was ˜0.33 μL, or roughly 20% that ofthe above monolithic column. Thus, on a column volume basis, the loadingcapacity of the PLOT column differed from the 200 μm i.d. monolithiccolumn by only a factor of 2 as a result of the long column length. Theloading capacities of the PLOT column prepared using 70% ethanoldecreased to ˜50 fmol for angiotensin I and ˜20 fmol for insulin. Thehigher percentage of ethanol resulted in the PS-DVB polymer phaseseparation occurring at an earlier stage of polymerization, likelyleading to less polymer coated on the tubing wall and thus a lowerloading capacity.

The ultimate goal of narrow bore LC-ESI-MS is to achieve low detectionlimits without sacrificing separation performance. The detection levelachievable with the PLOT column of the invention was evaluated using atryptic digest of bovine serum albumin (BSA). Ten attomoles of a BSAtryptic digest was bomb loaded directly onto the 10 μm i.d. PLOT columnand detected by the linear ion trap MS. Four peptides that provided goodMS/MS fragmentation and high SEQUEST scores were confidently identified,as shown in FIGS. 3A-3D. The extracted ion chromatogram of the peptide(YICDNQDTISSK) with the highest MS response (signal-to-noise ratio ˜210)is shown in the insert to FIG. 3B, indicating that the detection limitfor this peptide can, in principle, be in the hundreds of zmole range.

Example III Comprehensive Analysis of Large Complex Peptide Fragments ofEGFR

High sequence coverage proteomic analysis and comprehensivecharacterization of post-translational modifications at the trace levelare particularly important to help to address a variety of problems ofbiological interest. Often, one is faced with a limited amount of sampleand, yet it can be important to determine and quantitate individualprotein isoforms. An intermediate approach between top down and bottomup proteomics, extended range proteomic analysis (ERPA), was recentlyintroduced for comprehensive characterization of complex proteins⁴⁴.Lys-C was used as the proteolytic enzyme instead of trypsin, since theformer enzyme is a less frequent cutter. Thus, the complexity of thesample was reduced (˜2-3 fold lower number of peptide fragments than fortrypsin). The Lys-C digest, on average, led to longer peptides than thatfor the tryptic digest. In addition, extra arginines were frequentlyincluded in the digest fragment, leading to enhanced signal forpost-translationally modified peptides. Using this approach, greaterthan 95% sequence coverage was demonstrated in the analysis of aphosphorylated and glycosylated tyrosine kinase receptor, EGFR, at the75-fmole level using a 50-μm monolithic column⁴⁵.

Due to its open porous structure and high sensitivity features, PLOTcolumns according to the invention can also be effective for theanalysis of large post-translationally modified peptides, such as foundwith the ERPA approach. As a comparison with the earlier work, FIG. 4Ashows the base peak separation of ˜25 fmol Lys-C digest of EGFR on the4.2 m×10 μm i.d. PLOT column. On the basis of MS/MS analysis and tomanually match previously identified peptides⁴⁴, >70%, sequencecoverage, including post-translational modifications, was obtained. Asan example, FIG. 4B illustrates the MS/MS spectra of a large peptide, aswell as phosphorylated and glycosylated peptides and their extracted ionchromatograms. As seen in the figure, using a 45 min. linear gradient,symmetrical and narrow peaks were observed for the large peptide(w_(1/2), 7 s) (FIG. 4), phosphopeptide (w_(1/2), 9 s) (FIG. 4C), andglycopeptides (w_(1/2), ˜10s) (FIGS. 4D and 4E). The PLOT columndemonstrated high efficiency for the separation of all Lys-C digestpeptides of EGFR. The PLOT columns according to the invention should beeffective for even larger fragments, including intact proteins.

Example IV MicroSPE/nanoLC/ESI-MS Analyses

Successful practical operation of the PLOT column according to theinvention requires the ability to handle samples of at least of fewmicroliters volume. Since direct injection of such sample volumes on thePLOT column would take a long time, if successful at all, precolumnenrichment is an important procedure for sample handling. In addition,use of a precolumn would allow successful removal of salts and otherspecies in the sample solution that are deleterious to ESI-MS. Followingestablished procedure⁴⁶, the sample was pressure loaded manually on a 4cm×50 μm i.d. PS-DVB monolithic precolumn. Flows of ˜0.5 μL/min at apressure of 1000 psi were used for loading on the precolumn, such flowrates being ˜25× greater than that for direct loading on the PLOTcolumn. After loading, the precolumn was then inverted and butt-to-buttconnected to the PLOT column using a PicoClear union. An importantfeature of the union is that it can hold pressure up to 5000 psi. Thesample, loaded on the precolumn, was then back-flushed onto the PLOTcolumn. Automated loading of a precolumn and sample injection are alsowithin the invention.

The resolving power of the microSPE-nanoLC-ESI-MS system was evaluatedusing an in-gel tryptic digest of a proteomic sample of the archaeon, M.acetivorans. First, a 1 μL sample was loaded onto a 4 cm×50 μm i.d.PS-DVB monolithic precolumn using a pressure bomb at a flow rate of 0.5μL/min. FIG. 5A shows a 3.5-h gradient separation of only 4 ng of anin-gel tryptic digest sample of a gel fraction (>70 kDa) of M.acetivorans on the 4.2 m×10 μm i.d. PLOT column. The base peakchromatogram in FIG. 5A illustrates both the complexity of the sampleand the high resolving power of the system, with symmetrical peaks beingobserved throughout the entire separation. The peak capacity of thegradient separation using the PLOT column was estimated by examining theextracted ion chromatograms of individual components throughout theseparation window (FIG. 5B)⁴⁷. The 26 values of six high intensity peaksover the wide gradient range are between 0.21 and 0.30 s, leading to anestimation of peak capacity of ˜400. Even higher peak capacities areanticipated with full system optimization. A total of 689 uniquepeptides and 238 proteins (single-hit peptides excluded) were identifiedfrom this very small sample. The peptides and proteins were identifiedby automated searching of MS/MS spectra of the M. acetivorans database.The number of identified peptides and proteins increased significantly,from 689 and 238 to 1793 and 512, respectively, as the injection amountwas increased from 4 ng to 50 ng. Given that the sample was prepared byin-gel digest⁴⁷, 50 ng is still a relatively limited amount of material.

Finally, as a test the system of the invention, the five in-gel digestedfractions were combined together to simulate a global proteomic sample.For three repeat gradient runs from 150 ng of the combined in-gel digestof M. acetivorans, a total of 4409 unique peptides and 715 differentproteins (single hits excluded) were identified. These resultsdemonstrate the potential of the system of the invention for highresolution analysis with a limited sample amount.

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While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

1. A process for carrying out a chemical analysis method on a sample, said method comprising the steps of: providing a porous layer open tube (PLOT) capillary column, or channel in a microfabricated device, said column or channel comprising: a capillary column or channel having an i.d. of 15 μm or less, an entrance end and an exit end; a rigid porous layer separation medium comprising a highly crosslinked, macroporous, organic polymeric stationary phase layer attached to the inner wall surface of said column or channel, wherein said organic polymeric stationary phase layer comprises styrenic, methacrylic or acrylic monomeric units, or combinations thereof, wherein said organic polymeric stationary phase layer is from 0.5-3 μm in thickness, wherein said organic polymeric stationary phase layer is thermally stable to 250° C.; and wherein the reproducibility of retention time on comparable said columns or channels during use in liquid chromatography varies less than 10%; and an open unfilled bore inside said rigid porous layer, wherein said open bore is disposed around a central axis of said column or channel and extends the length of said column or channel; coupling a mass flow or concentration sensitive detector via an interface to the exit end of said column or channel; applying an aliquot of a liquid sample to said column or channel; conducting a liquid chromatographic separation procedure on said applied sample; and detecting said separated sample with said detector to carry out said chemical analysis method.
 2. The process of claim 1, wherein the reproducibility of retention time on comparable said columns or channels during use for said liquid chromatographic separation procedure varies less than 5%.
 3. The process of claim 1, wherein said liquid chromatographic separation procedure is conducted at a flow rate of 5-50 nL/min.
 4. The process of claim 1, wherein said column or channel during said liquid chromatographic separation procedure has a flow rate of 5-50 nL/min. at 6000 psi or less.
 5. The process of claim 1, wherein said column or channel has an i.d. of 10 μm or less.
 6. The process of claim 1, wherein said column has a length of greater than or equal to one meter.
 7. The process of claim 1, wherein said column has a length of greater than or equal to three meters.
 8. The process of claim 1, wherein, in said column or channel, said organic polymeric stationary phase layer attached to the inner wall surface of said column or channel comprises styrene and divinylbenzene monomer units.
 9. The process of claim 1, wherein, in said column or channel, said organic polymeric stationary phase layer attached to the inner wall surface of said column or channel comprises (C4-C18) alkyl methacrylate monomer units.
 10. The process of claim 1, wherein said detector is a mass spectrometer, a fluorescence detector, an electro-chemiluminescence detector or a nuclear magnetic resonance detector.
 11. The process of claim 1, wherein said interface is an electrospray ionization (ESI) interface or a matrix assisted laser desorption ionization (MALDI) interface.
 12. The process of claim 1, wherein said interface is an electrospray ionization (ESI) interface and said detector is a mass spectrometer.
 13. The process of claim 1, wherein said interface is a matrix assisted laser desorption ionization (MALDI) interface and said detector is a mass spectrometer.
 14. The process of claim 1, said process further comprising coupling a preparatory precolumn to the entrance end of said separation column or channel and applying said aliquot of said liquid sample to said preparatory precolumn.
 15. A porous layer open tube (PLOT) capillary column for liquid chromatography, said column comprising: a capillary column having an i.d. of 10 μm or less and a length of three meters or greater; a rigid porous layer separation medium comprising a highly crosslinked, macroporous, organic polymeric stationary phase layer attached to the inner wall surface of said column, wherein said organic polymeric stationary phase layer comprises styrenic, methacrylic or acrylic monomeric units, or combinations thereof, wherein said organic polymeric stationary phase layer is from 0.5-3 μm in thickness, wherein said organic polymeric stationary phase layer is thermally stable to 250° C.; and wherein the reproducibility of retention time on comparable said columns during use in liquid chromatography varies less than 10%; and an open unfilled bore inside said rigid porous layer, wherein said open bore is disposed around a central axis of said column and extends the length of said column.
 16. A capillary column according to claim 15, wherein the reproducibility of retention time on comparable said columns during use for liquid chromatography varies less than 5%.
 17. A capillary column according to claim 15, wherein, further, said column, during use for liquid chromatography, is capable of having a flow rate of 5-50 nL/min. at 6000 psi or less.
 18. A capillary column according to claim 15, wherein said organic polymeric stationary phase layer is poly(styrene-divinylbenzene).
 19. A capillary column according to claim 15, wherein said organic polymeric stationary phase layer comprises (C4-C18) alkyl methacrylate monomer units.
 20. A system for carrying out a chemical analysis method comprising: a porous layer open tube (PLOT) capillary column for liquid chromatography, said column comprising: a column having an i.d. of 10 μm or less, a length of three meters or greater, an entrance end and an exit end; a rigid porous layer separation medium comprising a highly crosslinked, macroporous, organic polymeric stationary phase layer attached covalently to the inner wall surface of said column, wherein said organic polymeric stationary phase layer comprises styrenic, methacrylic or acrylic monomeric units, or combinations thereof; wherein said organic polymeric stationary phase layer is from 0.5-3 μm in thickness; wherein said organic polymeric stationary phase layer is thermally stable to 250° C.; and wherein the reproducibility of retention time on comparable said columns during use varies less than 10%; and an open unfilled bore inside said rigid porous layer, wherein said open bore is disposed around a central axis of said column and extends the length of said column; and a mass flow or concentration sensitive detector coupled with an interface to the exit end of said column.
 21. The system of claim 20, wherein said detector is a mass spectrometer, a fluorescence detector, an electro-chemiluminescence detector or a nuclear magnetic resonance detector.
 22. The system of claim 20, wherein said interface is an electrospray ionization (ESI) interface or a matrix assisted laser desorption ionization (MALDI) interface.
 23. The system of claim 20, wherein said interface is an electrospray ionization (ESI) interface and said detector is a mass spectrometer.
 24. The system of claim 20, wherein said interface is a matrix assisted laser desorption ionization (MALDI) interface and said detector is a mass spectrometer.
 25. The system of claim 20, wherein said organic polymeric stationary phase layer attached to the inner wall surface of said column comprises styrene and divinylbenzene monomer units.
 26. The system of claim 20, wherein said organic polymeric stationary phase layer attached to the inner wall surface of said column comprises (C4-C18) alkyl methacrylate monomer units.
 27. The system of claim 20, wherein the reproducibility of retention time on comparable said columns during use for liquid chromatography varies less than 5%.
 28. The system of claim 20, wherein, further, said column during use for liquid chromatography is capable of having a flow rate of 5-20 nL/min at 6000 psi or less.
 29. The system of claim 20, said system further comprising a preparatory precolumn coupled to the entrance end of said separation column. 